Our laboratory investigates mechanisms of cell fate specification and patterning along the animal?vegetal (A?V) axis of the sea urchin (Strongylocentrotus purpuratus) embryo. Our major focus is to understand the gene regulatory networks and signaling pathways that specify ectodermal domains, which that derive from an undifferentiated pre-ectoderm and form the aboral, oral, neural and ciliagenic ectodermal territories. [unreadable] [unreadable] DEVELOPMENT OF RESOURCES AND TOOLS FOR MINING AND ANNOTATING THE SEA URCHIN GENOME. (33%; Zheng Wei, Staff Scientist). A major achievement was the creation of a gene list, using the gene prediction program, Genscan, which contributed to a composite gene list for the annotation of nearly 10,000 genes in the sea urchin genome. We used the gene predictions to design a whole-genome microarray for temporal profiling of mRNA expression at 5 embryonic stages, using 5 probes per gene on high density arrays (Nimblegen, Inc). The temporal profiles of a large number of well-studied genes have been confirmed and an expression database for about 28,000 different predicted genes is available to the research community for further studies. Several groups have expressed interest in using these microarrays as tools to characterize effects of experimental manipulations at the level of gene expression, and to identify additional genes in regulatory networks. [unreadable] [unreadable] MECHANISMS OF SPECIFICATION OF CELL FATES IN THE ANIMAL POLE DOMAIN (30%; Lynne Angerer, Senior Scientist; Shunsuke Yaguchi, Visiting Fellow). We are interested in understanding the gene regulatory networks underlying the initial specification of cell fates in the animal pole domain (APD) of the sea urchin embryo. This region is special because APD regulatory genes are refractory to repression by vegetal signals. The APD contains the precursors to the 6 serotonergic neurons of the embryonic nervous system and our specific goal is to identify genes constituting the core neurogenic gene regulatory network. The major approach this year has been to exploit the newly completed genome sequence to identify candidate genes encoding transcription factors that are expressed in neurogenic ectoderm. To date, 58 such genes have been identified in the sea urchin genome, 34 of which are expressed during embryogenesis in the APD, some exclusively in this region and some at unexpectedly early stages, well before gastrulation. Interestingly, four genes have been identified as encoding transcription factors that operate at the beginning of neural cell specification in a diverse set of organisms, including the coelenterates, an extremely ancient group in evolution, suggesting that these genes constitute part of the core neurogenic regulatory network of metazoa. The regulatory relationships among candidate neurogenic factors will be tested by blocking translation of each mRNA with morpholino antisense oligonucleotides (MASO) and assaying the consequent effects on expression of other genes using whole mount in situ hybridization and quantitative PCR. [unreadable] [unreadable] TGF-BETA SIGNALING IN ENDODERM DEVELOPMENT (33%; Aditya Sethi, Visiting Fellow). We have found that a TGF-beta signaling pathway is required for the development of early endoderm in the sea urchin embryo. Blocking this pathway with a small molecule inhibitor of the receptor, Alk4, results in significant delays and reduced expression of a specific set of early endoderm marker genes and in gastrulation. We have identified the ligand as activin B, the only TGF-beta, besides nodal, that functions through Alk4 and is expressed in the early embryo. Abrogation of activinB synthesis by either of two morpholino antisense oligonucleotides produces the same phenotype as does Alk4 inhibition, at both molecular and morphological levels. ActivinB signals are required in a subset of blastomeres fated to become endoderm, strongly suggesting that it is the long-sought early signaling pathway from underlying micromeres, which is thought to be one of the first signals in endoderm development downstream of beta-catenin nuclearization. [unreadable] [unreadable] SOXB1 AND NUCLEAR BETA-CATENIN CROSS-REGULATORY MECHANISMS (4%; Zheng Wei, Staff Scientist). SoxB1 is a key regulator of endomesoderm specification during late cleavage/early blastula stages. SoxB1 functions at the top of the endomesoderm gene regulatory network as an inhibitor of signaling through the canoniical Wnt signaling pathwy, by antagonizing nuclear beta-catenin. Conversely, beta-catenin, acting as a transcription cofactor, clears SoxB1 both by repression of transcription and, unexpectedly, through spatially regulated turnover of SoxB1 protein. We are testing whether mutual antagonism involves direct physical interaction between SoxB1 and beta?catenin. Yeast two-hybrid assays show that both full-length and the C-terminal half of SoxB1, required for beta-catenin-dependent SoxB1 turnover, can interact with sea urchin beta-catenin. Co-immunoprecipitation assays will test whether these proteins interact in the sea urchin embryo and may identify additional interacting proteins involved in critical early cross-regulation of SoxB1 and beta-catenin.